DESCRIPTION (Applicant's Abstract): Mechanical injury to the lung by high airway and vascular pressures cause increased vascular permeability and fluid extravasation in a number of disease states, but the segmental localization of vascular injury and cellular pathways are unknown. However, mechanical strain induced Ca2+ entry through stretch activated cation channels (SACC) appears to initiate endothelial cell (EC) retraction and adhesion release leading to vascular leak. We propose to establish SACC involvement in mechanical lung injury using three novel approaches with vertically integrated studies at the single channel, endothelial monolayer and intact lung levels. 1) Alveolar and extra-alveolar segmental permeabilities in isolated lungs will be assessed using segmental filtration coefficients (Kf) of arterial, venous an microvascular segments and electron microscopy under conditions which differentially alter segmental wall stresses. 2) Whole-cell voltage clamp and single-channel cell-attached patch clamp measurements of Ca2+ and monovalent cation entry currents evoked by cell deformation will be compared in cultured endothelial cells from rat pulmonary artery, vein and microvascular segments and Chinese Hamster Ovary (CHO) cells with and without transfection and expression of MID 1, a SACC gene product recently cloned from the yeast, Saccharomyces cerevisiae. 3) Pressure deformation induced increases in hydraulic conductance (Lp), decreases in protein reflection coefficient and changes in equivalent pore distribution in monolayers of the three EC phenotypes will be measured with and without expression of the MID1 gene product to determine the role of SACC in initiating the increased permeability as wet as certain cellular mechanism involved in the increase. Finally, the modulation of strain induced Ca2+ entry and permeability by cyclic nucleotides, cytoskeletal tone, cytokines and oxidants will be determined in the three experimental models to determine if polymodal gating and cellular feed back regulated SACC Ca2+ entry. At the end of this research we will unequivocally establish that SACC are critical for initiating strain induced vascular permeability increases and whether polymodal gating by cellular pathways and inflammatory mediators modulate this response to increase susceptibility to mechanical injury of patients with lung disease.